title: Regulation of mammalian autophagy by class II and III PI 3-kinases through PI3P synthesis.
abstract: Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for (~)35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.
authors.affiliation: Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, United States of America ; Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University Medical Center, New York, New York, United States of America.
authors.forename: Kelly
authors.initials: K
authors.lastname: Devereaux
authors.forename: Claudia
authors.initials: C
authors.lastname: Dall'Armi
authors.forename: Abel
authors.initials: A
authors.lastname: Alcazar-Roman
authors.forename: Yuta
authors.initials: Y
authors.lastname: Ogasawara
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhou
authors.forename: Fan
authors.initials: F
authors.lastname: Wang
authors.forename: Akitsugu
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Pietro
authors.initials: P
authors.lastname: De Camilli
authors.forename: Gilbert
authors.initials: G
authors.lastname: Di Paolo
mesh_terms: D000818:Animals; D001343:Autophagy / Q000502:physiology*; D002460:Cell Line; D058541:Class II Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D058546:Class III Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010588:Phagosomes / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011485:Protein Binding; D000071636:Protein Phosphatase 2C; D059748:Proteolysis
chemical_list: D018129:Phosphatidylinositol Phosphates; C055525:phosphatidylinositol 3-phosphate; D058541:Class II Phosphatidylinositol 3-Kinases; D058546:Class III Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C